For peginesatide, the first representative of a new generation of erythropoiesis-stimulating agents, a high misuse potential in elite sports is expected. Thus, the aim of the present thesis was the development of specific and sensitive mass spectrometry-based detection methods in various biological matrices relevant to doping analysis.
In the context of this background, the molecular structure of the active ingredient was identified before it was synthesised and characterised by means of mass spectrometric as well as gel electrophoretic techniques. As a mass spectrometric analysis of the intact analyte is not applicable for a specific detection approach in sports drug testing, the pegylated peptide was subjected to proteolytic digestion by the serine protease subtilisin. The thereby generated pentapeptide fragment allows for a sensitive detection by means of liquid chromatography coupled to tandem mass spectrometry and fulfils the required sequence coverage. Furthermore, its xenobiotic origin is supported by a non-natural amino acid present in the fragment.
Based on these results, detection assays for peginesatide in human blood (serum, plasma or dried blood spots) and urine as well as in horse serum were developed and validated in accordance with international guidelines. Proof-of-concept for the applicability of the methods was demonstrated by analysing dried blood spots, urine, and plasma samples from an in vivo study in rats, collected after a single therapeutic dose of peginesatide over a period of up to four days. In all three matrices, the characteristic pentapeptide fragment, obtained after proteolytic digestion, was unambiguously detected until the endpoint of the study, 72 h for dried blood spots and 96 h for urine and plasma specimens, respectively.
Thus, within the scope of preventive doping research, specific, sensitive, and valid detection methods are applicable to routine sports drug testing since the time of drug approval (March 2012). Moreover, they are readily transferable to other doping control laboratories and allow for the detection of an illicit application of peginesatide in different biological matrices relevant to doping analysis for at least several days.